J . gen. Microbioi. (1968), 51, 35 42
With 3 plates
Printed in Great Britain
35
Distinction between Bacterial and Algal Utilization of
Soluble Substances in the Sea
By A. L. S . M U N R O
Marine Laboratory, Aberdeen
T. D. BROCK
Department of Microbiology, Indiana University,
Bloomington, Indiana
AND
(Accepted for publication I September 1967)
SUMMARY
The presence of numerous bacteria and diatoms attached to the sand
grains of a littoral beach have been shown by fluorescence microscopy.
Bacteria and diatoms were found in a viable condition to depths exceeding
~ o c m .The rate of uptake of [14C]-acetate was measured over the range
10-5000,ug./l. and the results analysed by Michaelis-Menten kinetics.
By the use of autoradiography it was shown that the bacteria alone were
responsible for the uptake of PHI-acetate. It is concluded that algal heterotrophy is negligible in sea waters.
INTRODUCTION
The available substrate for heterotrophy in the sea comprise both particulate and
soluble organic material, the latter predominating by a factor of ten or more (Parsons
& Strickland, 1962). The soluble material is made up of heterogeneous compounds of
which only a fraction is likely to contain compounds of low molecular weight. Reliable reports of the concentration of individual compounds are few ; however, Siege1
& Degens (1966)have reported a mixture of 17free amino acids constituting 66 ,ug./l.
in Buzzards Bay, Cape Cod, and Degens, Reuter & Shaw (1964)reported glucose,
galactose and mannose concentrations of 3-18 pg./l. ( I O - ~ to IO-~M)in offshore
Californian seawaters. Wright & Hobbie (1965,1966)and Vaccaro & Jannasch (1966)
described the uptake of single substrates by pure cultures of bacteria at concentrations
in excess of I O - ~ M . In a similar survey of four pelagic species of algae, including one
known heterotroph, Sloan & Stickland (1966)concluded that uptake at concentrations
of 0.25 mg. C/l. (5 x IO-~M)was insufficient to balance even respiratory requirements.
The low concentration of suitable substrates dictates that measurement of heterotrophy within a natural population requires the development of special techniques.
In measuring the 'relative heterotrophic potential' of sea waters Parsons & Strickland
(1962)were the first to use a [14C]-labelled organic substrate in a manner analogous to
the [14C]-carbonatetechnique developed by Steeman Nielsen (1952) for marine photosynthesis. Both these authors and Wright & Hobbie (1965,1966)showed that the rate
of uptake of substrate by heterotrophic organisms within an aquatic population
followed a typical saturation curve which could be analysed by the Michaelis-Menten
equation at low concentrations (> IO-~M).At substrate concentrations < I O ~ M ,
3
'
2
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A. L. S. M U N R O A N D T. D. B R O C K
36
much higher than exist in natural waters Wright & Hobbie (1965, 1966) found that
both fresh water lake populations and pure cultures of algae isolated from these lakes
showed a second uptake mechanism based on diffusion kinetics and concluded that
the algae present in the natural population were responsible for most of the uptake.
Other authors (Rodhe, 1955; Wood, 1956) have concluded from direct observations
that algal cells in a natural community deprived of light do resort to heterotrophy.
The present paper sets out to measure the rate of uptake of a single organic substrate by a natural population and to determine if the algae present contributed to such
uptake.
METHODS
The experiments outlined below were made with naturally occurring microbial
populations attached to sand grains. Sand samples were taken from the littoral zone
of an exposed sandy bay in a sea loch, Loch Ewe, on the west of Scotland.
Uptake experiments. The methods used were essentially those described by Wright
& Hobbie (1965), adapted to suit sand populations. Experiments were made by incubating duplicate samples of 10 g. sand+25 ml. filtered (Whatman GF/C grade) seawater in 60 ml. Pyrex glass-stoppered bottles. Incubations were carried out at the prevailing sea-water temperature (10-14~) in dark incubators. High specific activity
uniformly labelled [l*C]-acetate (Radiochemical Centre, Amersham, Buckinghamshire) was prepared in sterile ampoules and added in pl. amounts at the start of incubation. Zero time controls were prepared by adding neutralized formalin immediately
after addition of the isotope. Samples were shaken twice during the course of I-hr
incubations. Metabolism was stopped by adding formalin, the sand filtered off and
washed with filtered sea-water. The sand samples were stored at - 15' and dried before counting on aluminium planchettes in an end-window counter of known efficiency.
Self absorption of radiation due to the sand was allowed for by multiplying counts/
min. by a factor of 21.5 (Baird & Wetzel, 1967).
Materialfor autoradiography. Sand samples were prepared by incubating 2 g. sand +
10 ml. filtered seawater in light or dark incubators with [14C]-carbonateor uniformly
labelled [3H]-acetate, respectively. Other conditions were as previously described
except that light incubations were for 5 hr at 1000ft.c. and at the end of incubation
all sand samples were washed with distilled water. Washed sand samples were treated
for 20-30 sec. at 20 kcyc./sec. in an ultrasonic disintegrator, then two drops of
supernatant fluid were spread immediately over the surface of a slide treated with
Ullrich's fixative, dried and washed.
Slides were dipped in Kodak NTB-2 liquid emulsion (diluted 112) held at 45", and
exposed for 3-7 days. Detailed methodology for the use of autoradiography with
micro-organisms in aquatic habitats will appear elsewhere (Brock & Brock, 1967). The
photographs of autoradiograms were taken on Polaroid Type 42 film with a Polaroid
MP-3 camera.
RESULTS
The microbial population. The microbial population attached to the sand grains in
question was found by fluorescent microscopy to be principally composed of diatoms
(Bacillariophyta; P1. I, fig. 1-3) and bacteria (Pl. 2, fig. 4-6). Samples were taken at
low tide where both populations extend to a depth of more than 15 cm. Additional
evidence of diatoms at these depths was shown by chlorophyll and chlorophyll
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Heterotrophy in the sea
37
phaeopigment analyses carried out on vertical profiles of the beach by Steel & Baird
(1967). The high ratios of chlorophyll to phaeopigment found by these authors as
well as uniform uptake of 14C0, throughout the profiles suggest algal cells in good
physiological condition. However, light penetration is only I % at 3 mm. depth in
quartz sand and studies of sand mixing indicate that only the top 5 cm. regularly
moves under wave action. Therefore much of the algal population may bein total
darkness for periods up to many months. The situation of these algae would seem
analogous to that described by other authors (Rodhe, 1955; Wood, 1956) who found
algal cells apparently in good physiological condition yet living in total darkness
under thick ice or at considerable depths in the oceans.
The bacterial population was readily detectable to a depth of 15 cm. as was shown
by viable counts of 0-5-3 x 105/g.sand and by staining the sand with acridine orange
(Pl. 2, fig. 4-6,). If the number of objects attached to the sand grains which fluoresce
green are truly all viable bacteria then the viable counts represent 0.1% or less of the
total population.
Kinetic analyses. An equation for uptake of a [14C]-substrateby an active population
was first proposed by Steeman Nielsen (1952) and further modified by Parsons &
Strickland (1962)
(1)
v =_
cf(Sn i-A_)
~
cpt,
where v is the velocity of substrate uptake (mg. 1.-l hr-l), c is the radioactivity contained in the population, Sn is the concentration (mg. I.-') of a given substrate present
in the natural sample and is assumed to be of negligible proportions compared to the
added substrate, A the concentration (mg. 1. -l) of added substrate, C the counts min.-l
from I pc [l4CC]in the counting assembly used, p the number of microcuries added to
the sample bottle, t the incubation time (hr), and f is a factor to compensate for any
discrimination between [14C] and [ W ] atoms, which in the present experiments is
neglected. In the present experiments c is the radioactivity associated with the sand
grains and is multiplied by a factor of 21.5 (Baird & Wetzel, 1967) to allow for selfabsorption of radiation by the sand grains and v, the velocity, is expressed as mg. g.-l
sand hr-l.
The results of measuring the velocity of [14C]uptake from uniformly labelled acetate
are shown from four different months in Fig. I. It can be seen that all the curves
resemble each other in sharply increasing velocities at low concentrations and thereafter by a decrease in rate. As all metabolism was terminated by addition of formalin
it is most likely that all the [14C]measured as uptake was no longer acetate but part of
the particulate fraction of the cells in question.
The rate of uptake (v) has been shown by Parsons & Strickland (1962) to follow a
saturation curve or Langmuir isotherm. If the uptake rate is proportional to time as
well it is possible to apply the Michaelis-Menten equation to elucidate some of the
properties of the natural population. Figure 2 shows that uptake at two substrate
concentrations was proportional to time over I to 1-5 hr periods.
Conversion of the velocity curve into a linear form using a modified LineweaverBurke equation gives
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A. L. S. M U N R O AND T. D. B R O C K
38
where v is the velocity of uptake at each concentration of substrate tested, V the
maximum rate attainable and Kt is a constant. Substituting from ( I ) gives
A
(31%
V'
Using equation (3) a plot of C,ut/c versus A can be used to solve for K,+Sn, the
intercept of the straight line on the x-axis and V, the inverse of the slope of the straight
line. The intercept on the ordinate, T .(hr), gives a measure of the turnover time of the
natural substrate (Wright & Hobbie, 1966).
C,ut-Kt+Sn
--C
v
+ -
;h
._
c,
'
$
c
(
0
400
800
1200
1600
2000 0
400
pg. added acetate carbon
800
1200
1600
2000
1.-l
Fig. I . Uptake velocity versus substrate concentration from four different months 1966.
Sand from 10cm. depth, from a low-water station. (a) 23 March at IO', (b) 18 May at I I',
(c) 27 July at 12-5",(d) 10 September at 14".
The result of plotting C,ut/cagainst a limited range of concentrations of A is shown
for the 4 months in Fig. 3. Only on two occasions, 18 May and 27 July, could all the
constants be satisfactorily solved. For 23 March and 10 September the straight line is
nearly parallel to the x-axis, implyingthat rate was proprotional to concentration. The
collected results for the parameters evaluated are shown in Table I. The values of V
were much greater than any of the reported results for water, a finding which can be
attributed to the much larger populations in a g. of sand than in 11. of sea water.
The second uptake mechanism described by Wright & Hobbie (1965, 1966) in
planktonic populations showed a linear increase in uptake velocity at increasing
substrate concentrations. The slope of this line, kd (hr-l), was found to be identical to
a similar constant derived from the kinetics of simple diffusion. On three occasions,
23 March, 27 July and 10 September, our results allowed the calculation of such constants, which are recorded in Table I .
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Heterotrophy in the sea
39
Wright & Hobbie (1966) attempted to distinguish kinetically the radioactive component taken up by algae as compared to bacteria. Autoradiography (Brock & Brock,
1966) provides a direct means of distinguishing bacterial from algal uptake. We have
2
4
8
6
Hr
Fig. 2. Uptake at A = 48 pg. and 0 = 848 pg. acetate-C over an 8 hr period on 22 March
1966. Temperature of incubation 10’.Sand from 10cm. depth from a low water station.
(4
40
40
30
20
n
0
30
0
20
-00
10
10
-.
0
‘z
\
50
L,
50
100
(4
20
(4
100
15
¶O
5
0
50
100
pg.
0
50
100
added acetate carbon 1.-1
Fig. 3. Data from Fig. I . plotted by modified Lineweaver-Burke equation showing
relationshipsbetween C pt/c and added acetate.
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A. L. S. MUNRO AND T. D. BROCK
40
prepared autoradiograms of material incubated in various concentrations of
[3H]-acetateand [3H]-glucose.We have also prepared autoradiograms of material incubated with 14C02in the light.
Table
I.
Collected kinetic data using naturally occurring microbial
populations attached to sand grains
Dates
Y (mg. acetate C x I O - ~ ):
Kt S (pg. acetate C) :
Tt (W
kd (hr x I O - ~ ) :
+
23. iii. 1966
36.5
18. v. 1966
80
170
21.5
-
27. vii. 1966
71'5
I0
1.5
26
10. ix. 1966
I22
At all concentrations of acetate or glucose, the diatoms were unlabelled, whereas
many labelled bacteria and bacterial microcolonies were seen. Plate 3, fig. 7 and 8
show two labelled bacterial clusters adjacent to unlabelled diatoms when a concentra.
was used, while P1. 3, fig. 9 and 10 show similar situations
tion of 3 3 0 , ~ g acetate/l.
when 990 and 330 ,ug.acetate/l. were used. The diatoms in the original preparations
were viable, as shown by the fact that they became labelled when incubated in the
light with 14C02.Two labelled diatoms are seen in P1. 3, fig. I I and 12.
The results illustrated in the photographs are representative of those seen in a large
number of microscope fields. In no case did any diatoms beome labelled when organic
substrates were used.
DISCUSSION
From these results we can conclude that the assimilation of organic materials was by
the bacteria associated with the sand, even when high concentrations were used. We
assume that the bacteria stripped from the sand grains by brief ultrasonic treatment,
and hence detected autoradiographically, were typical of those seen by fluorescence
microscopy attached to the sand. The large numbers of bacteria seen on the sand
particles should be emphasized. Only by reflected fluorescence microscopy can these
organisms be readily seen, and virtually every sand particle observed was well
colonized. Diatoms were also present on most sand particles, but seemed to be present
in lower numbers than bacteria. However, Steel & Baird (1967) found a high correlation between the chlorophyll and carbon content of this sand, suggesting that the algal
biomass constituted most of the organic material present.
By use of the Wright & Hobbie technique it was possible to calculate diffusion
constants. From this and the evidence of a large algal biomass it might have been
concluded that uptake of organic substances at higher concentrations was algal. Autoradiography shows that this conclusion is not justified, and emphasizes the danger of
attempting to determine the presence of two components of a process by kinetic data
alone. The reason for altered kinetics at higher substrate concentrations is obscure,
but autoradiography shows that bacteria were responsible for all the uptake measured
and hence the shape of the velocity versus concentration curves. On two of the four
occasions it was possible to evaluate V and Kt + Sn suggesting kinetics compatible
with the Michaelis-Menten equation. On the other two occasions Fig. 3a, d show a
zero-order reaction, a result also found by Wright & Hobbie (1965) for some natural
populations. The reasons for these findings remain obscure.
In aquatic environments organic concentrations are generally low, although in the
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Heterotrophy in the sea
41
present case absorption phenomena may have led to concentrations on the sand
higher than those in the free water. Bacteria are clearly able to compete successfully
for the limited organic matter available, and it seems reasonable to generalize this
conclusion to other aquatic situations. Aquatic bacteria have undoubtedly evolved for
growth on low organic concentrations, and it seems likely that any small molecular
weight organic substances which become available in the environment will be preferentially assimilated by them.
The diatoms living below the photic zone are viable as shown by autoradiography of
cells exposed to [14C]-carbonatein the light and also by the results of Steel & Baird
( I 967) on [14C]measurements on vertical profiles. The viability of these diatoms cannot be explained by heterotrophy. A parallel between this environment and finding
algae in deep oceanic waters suggests that a low level of endogenous metabolism may
play an important part in the survival of algal cells which had otherwise been assumed
to resort to heterotrophy. In closing, we might point out that autoradiography is a
powerful tool in the study of energy and trophic relations in microbial ecosystems.
We wish to acknowledge the indispensable assistance of M. Louise Brock in the
autoradiography experiments. T. D. Brock is a Research Career Development Awardee
of the U.S. Public Health Service (AI-K3-18, 403), and his research is supported in
part by a grant from the U.S. National Science Foundation (GB-5258).
REFERENCES
BAIRD,I. E. & WETZEL,
R. G. (1967). Method for the determination of zero thickness activity of 14C
labelled benthic algae in sand counted by end window geiger counter. Limnol. Oceanogr. (in
Press).
BROCK,T. D. & BROCK,M. L. (1966). Autoradiography as a tool in microbial ecology. Nature,
Lond. 209,734.
BROCK,M. L. & BROCK,
T. D . (1967). The application of auto-radiographic techniques to ecological
studies. Mitt. int. Verein. theor. angew. Limnol. (in Press).
DEGENS,
E. T., REUTER,
J. H. & SHAW,N. F. (1964). Biochemical compounds in offshore California
sediments and seawaters. Geochim. cosmochim. Acta 28, 45.
PARSONS,
T. R. & STRICKLAND,
J. D. H. (1962). On the production of particulate organic carbon by
heterotrophic processes in sea-water. Deep Sea Res. 8, 2 I I .
RODHE,W. (1955). Can plankton production proceed during winter darkness in subarctic lakes?
Verh. int. Verein. theor. angew. Limnol. 12, 117.
SIEGEL,
A. & DEGENS,
E. T. (1966). Concentration of dissolved amino acids from saline waters by
ligand-exchange chromatography. Science, N. Y. 151, 1098.
SLOAN,P. R. & STRICKLAND,
J. D. H. (1966). Heterotrophy of four marine phytoplankters at low
substrate concentrations. J . Phycol. 2, 29.
STEEL,
J. H. & BAIRD,I. E. (1967). Production ecology of a sandy beach. Limnol. Oceanogr. (in Press).
STEEMAN,
NIELSEN,
E. (1952). The use of radioactive carbon for measuring organic production in the
sea. J . Cons.perm. int. Explor. Mer 18, I 17.
VACCARO,
R. F. & JANNASCH,
H. W. (1966). Studies on heterotrophic activity in seawater based on
glucose assimilation. Limnol. Oceanogr. 11, 596.
WOOD,E. J. F. (1956). Diatoms in the ocean deeps. Pacif. Sci. 10, 337.
J. E. (1965). The uptake of organic solutes in lake water. Limnol. Oceanogr.
WRIGHT,
R. T. & HOBBIE,
10, 22.
WRIGHT,R. T. & HOBBIE,
J. E. (1966). Use of glucose and acetate by bacteria and algae in aquatic
ecosystems. Ecology 47, 447.
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42
A. L. S. M U N R O A N D T. D. B R O C K
EXPLANATION O F PLATES
PLATEI
Figs. 1-3. Transmitted ultraviolet illumination showing fluorescing chloroplasts within diatoms.
Fig. I. A sand grain from 8 to 10cm depth. 2 May 1966.Sample taken at a low-water station.
Fig. 2. Sand grains from o to I cm depth. 2 May 1966.Sample from sublittoral beach.
Fig. 3. Sand grains from 8 to 10cm depth. 5 July 1966.Sample taken at a low-water station.
PLATE2
Figs. 4-6. Incident ultraviolet illumination with a Carl Zeiss microscope oil immersion N.A. 1.3.
Surfaces of sand grains from low water station, stained with acridine orange ( I /62,000yo,w/v) showing numerous bacteria and also diatoms. 4 September 1966.
PLATE3
Figs. 7-12.Photographed with a Carl Zeiss Phase microscope, oil immersion N.A. 1-25 unstained.
All photographs at same magnification.
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Jourrial of General Microbiology, Vol. 51,No.
A. L. S . MUNRO
AND
I
T. D. BROCK
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Plate I
(Facing p . 42)
Jotiriial of General Microbiology, Vol. 5 I , No. I
A. L. S . MUNRO
AND
T.D. BROCK
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Plate
2
Journal of General Microbiology, Vol. 51, No.
A . L. S. M U N R O
AND
I
T. D. BROCK
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Plate 3